Project Summary RBC transfusion can induce alloantibodies that can make it difficult to find compatible blood for future transfusions, increase the likelihood of incompatible transfusion reactions, and directly increase morbidity and mortality in patients with sickle cell disease (SCD). Empirical data have shown that although antigen-matching protocols reduce RBC alloimmunization, no currently available strategies completely prevent RBC-induced alloantibody formation following therapeutic transfusion. This in part reflects a lack of understanding regarding key factors responsible for initiating RBC alloimmunization. Our central hypothesis is that marginal zone (MZ) B cells represent a central node in the initiation and orchestration of immune responses to RBC alloantigens, and thus may be a viable immunological target for the development of novel strategies to not only predict, but also prevent RBC alloantibody production. Our hypothesis is formulated on the basis of our recent discovery that MZ B cells are required for the development of RBC alloantibody formation in a pre-clinical model. As MZ B cells can traffic antigen to B cell follicles, the requirement of MZ B cells in RBC alloimmunization may reflect the trapping and subsequent delivery of allogeneic RBCs to B cell follicles, where antigenic substrate is then used by CD4 T follicular helper cells (TFH) to drive effective germinal center reactions responsible for significant alloantibody formation. As antigen trapping requires B cell receptor engagement and MZ B cells possess a restricted and distinct repertoire of antibody specificities in any given individual, these data also suggest that differences in the precursor frequency of RBC alloantigen specific MZ B cells may predict the likelihood that an individual will develop alloantibodies following RBC alloantigen exposure. Furthermore, as toll-like receptor (TLR) agonists can directly increase MZ B cell activity, conditions that directly engage TLRs, such as excess hemin released during acute chest syndrome (ACS) in patients with SCD, may increase the likelihood of RBC alloimmunization by positively impacting MZ B cell function, and therefore may explain in part the increased propensity of patients with SCD to develop RBC alloantibodies. These preliminary data identify MZ B cells as key players in regulating RBC alloantibody formation, and in so doing provide a unique opportunity to define the role of MZ B cells in predicting and preventing RBC alloimmunization. To accomplish this, Project 2 will use a complementary approach of clinical and pre-clinical studies to weld observational data with mechanistic studies in order to define the role of MZ B cells in the regulation of RBC alloimmunization by testing the following specific aims: Aim 1: Define the role of MZ B cells in RBC alloimmunization. Aim 2: Define the impact of ACS on MZ B cell and TFH activation. We believe that successful completion of these aims not only possess the capacity to provide new mechanistic insight into key aspects of RBC alloimmunization, but may also establish an important framework to develop rational approaches designed to prophylactically predict and ultimately prevent RBC alloantibody formation in chronically transfused individuals.